Change MGI:1891436 Foxp3 coTF annotation to dbTF

[RLovering]

Hi Harold

Foxp3 is known to be a DNA binding transcription factor. I looked at this paper (PMID:15790681) and the data does look like this supports the coTF annotation, however Colin confirmed that although this looks like Foxp3 is both a dbTF and coTF, https://pubmed.ncbi.nlm.nih.gov/15790681/ does not provide sufficient evidence to support the coTF annotation.

I looked for papers that have cited this one and https://onlinelibrary.wiley.com/doi/full/10.1111/imm.13178 confirms the view that Foxp3 is binding DNA to repress expression (see the subsection NFAT and AP‐1

Foxp3 binds NFAT and inhibits its transcriptional activities.27, 28 Wu et al. analysed the crystal structure of NFATc2 (NFAT1), the forkhead domain of FOXP2 (which is considerably similar to that of FOXP3) and a 19 base‐pair‐DNA sequence from the IL2 promoter (namely, ARRE2), and characterized the NFATc2:FOXP2:DNA complex. Interestingly, when making a complex with NFATc2, the forkhead domain bound the DNA sequence that are occupied by Fos and Jun when they bind NFAT, suggesting that Foxp3 competitively inhibit the NFATc2:Fos:Jun complex by binding NFATc2 and replacing Fos and Jun.27 However, this view is challenged, as another Foxp member, Foxp1, is constitutively expressed by CD4+ T‐cells,67 which means that Foxp1 may constitutively make a complex with NFAT in the absence of Foxp3.

Please could you review this annotation and consider changing these to GO:0000981 | DNA-binding transcription factor activity, RNA polymerase II-specific or deleting the annotation

MGI | Q99JB6 | Foxp3 | enables | GO:0003714 | transcription corepressor activity | ECO:0000314 (IDA) |   | PMID:15790681

Many thanks

Ruth

[krchristie]

Foxp3 is known to be a DNA binding transcription factor. I looked at this paper (PMID:15790681) and the data does look like this supports the coTF annotation, however Colin confirmed that although this looks like Foxp3 is both a dbTF and coTF, https://pubmed.ncbi.nlm.nih.gov/15790681/ does not provide sufficient evidence to support the coTF annotation.

What exactly do you feel is the problem with https://pubmed.ncbi.nlm.nih.gov/15790681/ and the coTF annotation? This paper definitely does NOT support making a dbTF annotation.

I looked for papers that have cited this one and https://onlinelibrary.wiley.com/doi/full/10.1111/imm.13178 confirms the view that Foxp3 is binding DNA to repress expression (see the subsection NFAT and AP‐1

Here is the entire subsection you refer to:

NFAT and AP‐1

The AP‐1 transcription factors are formed by heterodimerization of bZIP proteins, including Jun (e.g. c‐Jun, JunB and JunD), Fos (e.g. c‐Fos) and ATF (e.g. ATF2, BATF).65 Particularly, Fos and Jun regulate TCR signal downstream genes.65 Foxp3 is proposed to bind c‐Jun and inhibit AP‐1 activities,66 although the evidence is limited.

Foxp3 binds NFAT and inhibits its transcriptional activities.27, 28 Wu et al. analysed the crystal structure of NFATc2 (NFAT1), the forkhead domain of FOXP2 (which is considerably similar to that of FOXP3) and a 19 base‐pair‐DNA sequence from the IL2 promoter (namely, ARRE2), and characterized the NFATc2:FOXP2:DNA complex. Interestingly, when making a complex with NFATc2, the forkhead domain bound the DNA sequence that are occupied by Fos and Jun when they bind NFAT, suggesting that Foxp3 competitively inhibit the NFATc2:Fos:Jun complex by binding NFATc2 and replacing Fos and Jun.27 However, this view is challenged, as another Foxp member, Foxp1, is constitutively expressed by CD4+ T‐cells,67 which means that Foxp1 may constitutively make a complex with NFAT in the absence of Foxp3.

Functional significance of the Foxp3−NFAT interaction is still not fully clear. Wu et al. showed that Foxp3 mutants that did not bind NFATc2 were defective in mediating Il2 repression, the upregulaton of Treg‐associated surface proteins (e.g. CD25) and suppressive activities,27 suggesting that the Foxp3−NFATc2 complex regulates transcriptional activities of a wide range of genes. The Foxp3‐binding domain of NFATc2 is conserved with other NFAT isoforms (i.e. NFAT2, NFAT3 and NFAT4).27 However, it is not known whether different NFAT isoforms play different roles in regulating Foxp3 function.

@LiNiMGI and I do not agree that this text makes a definitive statement that the only role of Foxp3 is to be a dbTF.

Please could you review this annotation and consider changing these to GO:0000981 | DNA-binding transcription factor activity, RNA polymerase II-specific or deleting the annotation

MGI | Q99JB6 | Foxp3 | enables | GO:0003714 | transcription corepressor activity | ECO:0000314 (IDA) | | PMID:15790681

At the moment, we are inclined to leave this annotation as is.

[RLovering]

Hi Karen

thanks for looking at this further. We did not complete a review of all the literature. I am passing this to Colin in case he has any additional comments to make. We will be including this protein as a dbTF but there are dbTFs with coTF or other functions

best

Ruth

[krchristie]

Hi Ruth,

It totally makes sense to include it as a dbTF. We'll take a look again if Colin as any further suggestions.

Thanks,

-Karen

Hi Karen

thanks for looking at this further. We did not complete a review of all the literature. I am passing this to Colin in case he has any additional comments to make. We will be including this protein as a dbTF but there are dbTFs with coTF or other functions

best

Ruth

[colinlog]

Dear All, I think the following on this matter: Competing for DNA binding sites is not a co-repressor activity in the sense that it is not a MF that is performed by the protein in order to assist a dbTF that is bound to a DNA sequence to effect its activator or repressor function. These are more simply dbTFs that compete for the same site. It would already be going out on a limb to state that FOXP2 is an inhibitor of the other dbTFs, so I would not characterise its MF as such. In this case FOXP2 appears to me to behave as a dbTF that represses the activity of a regulatory DNA element by binding to it. That is dbTF MF, not co-repressor MF.

[RLovering]

Hi Karen

are you going to remove the coTF annotation? or leave as it is? It would be good to get this ticket closed, ideally with the coTF annotation removed Thanks Ruth

[krchristie]

Hi Karen

are you going to remove the coTF annotation? or leave as it is? It would be good to get this ticket closed, ideally with the coTF annotation removed Thanks Ruth

Hi Ruth,

With various other responsibilities in the mix, I haven't had a chance to recheck the paper and decide if I agree with @colinlog to decide if we feel it is correct to delete the coTF annotation or not. I will try to get to this next week.

Thanks,

-Karen

[RLovering]

Hi Karen we have only about 20 human proteins still annotated as dbTFs which shouldn't be. 7 are due to Ensembl annotations from the mouse ortholog. It would be great if these could get fixed soon. Apologies if you have already done these, and they just haven't shown up in Protein2GO yet. I will add this text to all of the outstanding tickets if I can find them. Hope all is well with you Ruth

[colinlog]

Dear all, I looked-up a review that attempts to clarify the molecular functions of human FOXP3 splice isoforms (PMID:29593749). This 2018 review concludes that (i) there is a Forkhead DNA binding domain in all FOXP3 splice-soforms and (ii) that the splice isoforms compete for co-factors. They also cite reference 69 Hossain DM, Panda AK, Manna A, Mohanty S, Bhattacharjee P, Bhattacharyya S, et al. FoxP3 acts as a cotranscription factor with STAT3 in tumor-induced regulatory T cells. Immunity (2013) 39:1057–69. doi:10.1016/j.immuni.2013.11.005 . That is about a co-factor function for FOXP3, just like the 2005 PNAS paper at https://pubmed.ncbi.nlm.nih.gov/15790681/.

The 2005 paper https://pubmed.ncbi.nlm.nih.gov/15790681/ that is brought-up above by Karen, does not exclude that the DNA bound form of FOXP3 is the active form. In other words, these researchers use a vague concept for co-factor, namely that two dbTFs cooperate or antagonize each other and thus are cofactors for each other. Following the current guidelines, as FOXP3 is not demonstrated in that paper to not need its DNA binding domain to bind DNA to exert its function at the cytokine genes it should be considered to be acting as a dbTF. That is the issue I have with concluding from https://pubmed.ncbi.nlm.nih.gov/15790681/ that FOXP3 has coactivator or corepressor activity "over and above" its dbTF activity and that of the cofactor activities that DNA-bound FOXP3 recruits.

By contrast, reference 69, a paper from 2013, has experiments that would indicate that the Forkhead DNA binding domain of FOXP3 is not required for it to bind to STAT3 and to co-activate its target gene IL10. I personally do not like the fact that this is transient overexpression-based experimentation which, together with the physiological level-competition for co-factors that has been documented between FOXP3 splice-isoforms, can be used to argue that the results of reference 69 are overexpression artefacts related to squelching phenomena, the fact that the overexpressed protein when in the nucleus competes for protein-binding partners of the DNA bound dbTFs. Still, 'the literature' does indeed state that FOXP3 has coactivator activity (reference 69 / PMID:24315995 / Hossain DM et al. 2-13). FOXP3 may thus be annotated. I am personally not convinced this is true. I am inclined, to favor models such as those described by Karen on August 7 2020 here above, where the DNA binding activity is part of the action mechanism, the molecular activity, of FOXP3. But that is an opinion. The 2013 Hossain DM paper fuels the proposition that FOXP3 is also a co-factor that cooperates with STAT3 without a need for its DNA binding domain. I note that their model involves recruitment of the HAT1 Histone H4K5 and H4K12 acetyltransferase, which is classically known as a nucleosome deposition/maturation enzyme during DNA replication and repair, rather than as a transcription regulatory enzyme such as the TIP60 coactivator that targets the same lysines. Notwithstanding this shortcoming in the 2013 paper I have jotted down below some thoughts on the matter of FOXP3 being a coactivator or not.

It is certain that dbTFs can compete for DNA binding sites and be mutually exclusive though this competition. However, they may also cooperate by concomitantly binding to adjacent DNA binding sites and in that sense cooperate. Binding to other dbTFs is part of this DNA-sequence directed dbTF cooperation. That does not make them co-factors if the definition for co-factors is that they are recruited to promoters and enhancers by DNA-bound DNA binding transcription factors. To be absolutely clear, defining the dbTF and co-factor activities in the current way does not preclude that dbTFS cooperate or antagonise each other. Even by binding to each other, as appears to be the case for FOXP3 and STAT3.

In fine, the fact that we have stated that dbTFs provide the genomic addresses where the regulation takes place, whilst cofactors do not do that but are merely recruited or locally activated by the DNA-bound dbTFs is but the reflection of a working model. Reality will indicate how often this model is ambiguous or even wrong; where the so-called cofactors do contribute genomic localisation information and where the so-called dbTFs also regulate transcription without binding specific DNA sequences. However, in the present case, to my knowledge, FOXP3 has not been demonstrated to be recruited by STAT3 to most of its DNA bound sites. Only for IL10. The authors do show an indirect assay that shows that silencing of FOXP3 or STAT3 has similar effects on a number of genes (IL6, VEGFA, CMYC,BCL2L1, and CCND1) but not at that of the TGFb1 promoter. Hence, it would be a misnomer to say that FOXP3 a STAT3 co-factor at all these genes since alternative interpretations of the experiments of Hossein DM and colleagues are still in order. As there are some 350 non-review papers that have FOXP3 and STAT3 in the text, and as there are papers stating that STAT3 induces FOXP3 transcription T-cells as well as papers that claim that STAT3 epigenetically silences the FOXP3 promoter in cancer cells, it looks like this is a field where cell-type specific details are very very important. Will the association of the STAT3-cofactor MF to FOXP3 in tumour-associated mouse T-cells help to clarify the situation? Time will tell.